Project Summary/Abstract Antibiotic resistance threatens to return our society to a pre-antibiotic era. Evolutionary theory predicts that when bacteria mutate to become antibiotic resistant, they should become less fit than their antibiotic susceptible relatives when antibiotics are absent. Surprisingly, recent findings suggest that certain resistance mutations can increase bacterial fitness and virulence. This suggests that treatment could potentially eliminate susceptible isolates, only to have them replaced by hypervirulent, antibiotic resistant siblings. Despite the potential dire consequences of these virulence enhancing resistance mutations (VERMs), little is known about how VERMs enhance virulence nor how frequently VERMs arise during infection. The specific aims of this proposal are to determine 1) the mechanisms by which VERMs enhance virulence 2) how frequently VERMs arise during chronic infection and whether they are associated with poor patient outcomes. First, this proposal will determine how two different VERMs enhance virulence in hypervirulent, aztreonam resistant Pseudomonas aeruginosa strains. Because the two VERMs each affect genes that normally inhibit aztreonam efflux pump expression, the first aim of this proposal will test the hypothesis that increased aztreonam efflux pump expression leads to increased virulence. Mutant bacteria will be constructed and tested in a murine lung infection to determine if increased efflux expression is both necessary and/or sufficient to increase virulence. This aim will also use transcriptomics to determine all of the genes regulated by the two genes affected by the VERMs, including any virulence genes they may regulate. The second aim will determine the frequency of VERMs in clinical isolates. This will be accomplished using whole genome sequencing to identify aztreonam resistance mutations in P. aeruginosa collected from cystic fibrosis patients. Then the most commonly encountered resistance mutations will be tested for their effects on virulence using the acute lung infection model. Finally, isolates collected from a clinical observational study will be analyzed to determine if the identified VERMs are more commonly encountered in patients that experienced pulmonary exacerbations than in patients that that did not experience exacerbations. The long-term objectives of this project are directly relevant to the NIAID mission to better understand infectious diseases, and in doing so help guide the development of new diagnostic tools and therapeutics for antibiotic resistant bacterial infections.